Cone cells are responsible for photopic vision, the visual process under normal light conditions. The cone receptors must operate over a wide range of light intensities and cover the full range of the visible spectrum. The ability to function under these diverse conditions is due primarily to the highly optimized GPCR light-transducing proteins informally called cone pigments. These proteins have absorption maxima that range from 350 to 660 nm, and upon the absorption of light, undergo an efficient photobleaching sequence to produce an activated protein. Subsequent binding of transducin to the activated protein results in a nerve impulse and vision. A key observation made during the previous NIH funded study was that cone pigments undergo a counterion switch during photoactivation. A key aim of this study is to explore whether a counterion switch mechanism is also active in the red and blue cone pigments, and if so, to characterize the molecular details. To achieve this goal, we will use vibrational and electronic spectroscopy at temperatures from 10K to ambient to trap and characterize the photobleaching intermediates. Site directed mutagenesis will be used to identify the key residues responsible for wavelength selection and the nature of the counterion switch. It is clear from homology studies that many of the red cones differ from the green, blue and UV cones in nature and implementation of the counterion switch. Indeed, it is possible that the red cones lack this mechanistic feature entirely. An additional aim of this study is to systematically identify the mechanisms of wavelength selection in the UV, blue, green and red cones. Although our research identified key features of wavelength selection in the blue and UV cones, much remains to be understood. Our inclusion of the red cones in this study is new, and our enthusiasm for this topic rests in part on our belief that the red cones are fundamentally different. We have preliminary evidence, presented in our preliminary studies discussion, that the deep red cones use at least one new mechanism for wavelength selection involving manipulation of the chromophore ring conformation. The combination of unique wavelength selection and a significantly different (or absent) counterion switching mechanism make the red cones an important target. Our studies will include the use of molecular orbital theory to probe structure-function relationships in the cone pigments, and to calculate the spectroscopic properties of the bound chromophores. We will refactor our MNDO-PSDCI code and improve the interface to make these procedures more useful to the scientific community. As before, we will provide these procedures to interested researchers without charge. PUBLIC HEALTH RELEVANCE: There is a growing need to understand the photobleaching and recovery mechanisms associated with light exposure in cone photoreceptors. Because these cells are essential for human photopic vision, it is important to understand the structure and function relationships in the associated light transducing pigments. The project goals of this research may help understand macular disease, which involves loss of cone cells, cone dystrophy, and other eye diseases, which involve damage or diminished function of retinal photoreceptors.